All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Plant pathogens challenge efforts to maximize crop production through their ability to rapidly develop resistance to pesticides, which can result in immense yield losses on an annual basis. One of the main research goals today involves the development of new tools to control pathogens.
Fungal biocontrol agents have become an important alternative to the use of chemicals due to environmental concerns. Biological control can be achieved by one or a combination of mechanisms: antibiosis, mycoparasitism, competition and induced resistance in the host plant. These mechanisms can hinder growth and development of the pathogen, thereby reducing disease. The complex mode of action of biocontrol agents reduces the ability of the pathogens to develop resistance. The development of a biocontrol agent starts with the discovery of antagonists, followed by isolation and characterization of their potential biocontrol activity.
A few biofungicidal products are commercially available in some countries, for example AQ10, which contains conidia of Ampelomyces quisqualis, and Sporodex, which is based on conidia of the yeast Pseudozyma flocculosa, both used for the control of powdery mildew. There are also some products based on Trichoderma spp., such as Throcodex and Thrichopel, which are used against gray mold, root rot and root wilt. However, the use of biological control is still only moderate relative to that of chemical fungicides [Paulitz, T. C. and Belanger, R. R. (2001) Annu Rev. Phytopathol. 39:103-133].
Epiphytic yeasts colonizing different plant surfaces are thought to have biocontrol activity and to provide a natural barrier against some plant pathogens [Avis, T. J. and Belanger, R. R. (2001) Appl. Environ. Microbiol. 67(2):956-960; Urquhart, E. J. and Punja, Z. K. (2002) Can. J. Microbiol. 48(3):219-229]. Biocontrol activity of yeasts and yeast-like fungi has been demonstrated for postharvest diseases [Spadaro, D., and Gullino, M. L. (2004) International Journal of Food Microbiology 91:185-194] and diseases in the greenhouse [Paulitz, T. C. and Belanger, R. R. (2001) Annu Rev. Phytopathol. 39(103-133)]. Pseudozyma spp. are a small group of yeast related to the Ustilaginales [Boekhout, T. (1995) General and Applied Microbiology 41(359-366)]. They are mostly epiphytic (derive moisture and nutrients from the air and rain) or saprophytic (grow on and derive their nourishment from dead or decaying organic matter), and they are non-pathogenic to plants and animals [Avis, T. J. and Belanger, R. R. (2002) FEMS Yeast Res 2(1):5-8]. Pseudozyma rugulosa and P. flocculosa have recently been found to exhibit biological activity against the different powdery mildews with which they are associated [Dik, A. J., et al. (1998) Eur. J. Plant Pathol. 104(413-423]. P. flocculosa has been found to secrete an unusual fatty acid that displays antibiotic activity against several pathogens [Avis, T. J. and Belanger, R. R. (2001) Appl Environ Microbiol 67(2):956-960; Avis, T. J., et al., (2001) Phytopathology 91(3):249-254;]. On the other hand, Avis et al. [Avis, T. J., et al., (2001) Phytopathology 91(3):249-254] found no colony collapse of powdery mildew (Sphaerotheca fuliginea (Schlechtend.:Fr.) Pollacci) and no production of antifungal fatty acids by Pseudozyma aphidis isolated from aphid secretions (isolate CBS 517.83). P. aphidis is a close relative of P. rugulosa [Begerow, D. and Bauer, R. (2000) Mycol. Res. 104(53-60)], which was first isolated from aphid secretions [Henninger, W. and Windisch, S. (1975) Arch. Microbiol. 105(1):47-48] but has also been found on plant surfaces [Allen, T. W., et al., (2004) Can. J. Microbiol. 50(10):853-860].
The inventors recently isolated the epiphytic yeast Pseudozyma aphidis (isolate L12) from strawberry leaves. Isolate L12 was associated with the collapse of powder mildew colonies. Data presented herein demonstrates that L12 secretes extracellular metabolites which inhibit several fungal and bacterial pathogens in vitro. In addition, application of L12 spores on detached tomato leaves or whole tomato plants in the greenhouse significantly reduced Botrytis cinerea infection. The inventors therefore further characterize the L12 isolate of P. aphidis, developing it as an efficient biocontrol agent against plant pathogens. The conditions needed for mass production of active culture are characterized by testing various temperatures and media and monitoring spore concentration and activity by B. cinerea bioassays. The establishment and spread of P. aphidis on the host plant using microscopy is assayed. The inventors also assay the secreted fraction of L12 against various pathogens in vitro. In addition, P. aphidis L12 spores are applied on tomato plants in the greenhouse and their ability to control fungal and bacterial pathogens in vivo is verified. The new and efficient biocontrol agents provided by the invention may thus contribute to reducing the amount of chemicals required for pathogen control, and as such can genuinely benefit farmers, consumers and the environment.
The inventors herein develop the practical application of P. aphidis L12 as a biocontrol agent based on naturally-occurring fungi that increase plant resistance to fungal, viral, bacterial, and insect infestations as well as enhancing growth. The presented results demonstrate the high potential of P. aphidis L12 for the control of fungal and bacterial plant pathogens that cause major damage to crop plants. Furthermore, the novelty of the isolate is that it is easy to produce, very stable and effective in low concentration. Chemicals that have traditionally been used to control food plant pathogens are being banned or are no longer effective and organic farmers are not allowed to use them. Other control strategies are either unavailable or impracticable. Driven by environmental concerns and the growing demand for organic products, there is a pressing need for the development of new biological defense strategies.
Thus, one object of the invention is the provision of a composition comprising Pseudozyma aphidis cells, components or products, providing improved plant resistance to pathogenic infection. Moreover, the invention further provides compositions for the treatment, amelioration, inhibition or elimination of an established infection or infestation.
Another object of the invention is the provision of a composition comprising Pseudozyma aphidis cells, components or products, for the induction of plant immune response.
Yet another object of the invention is the provision of a composition comprising Pseudozyma aphidis cells, components or products, for improving plant growth and yield.
These and other objects of the invention will become apparent as the description proceeds.